We have characterized CHO cell mutants with temperature-sensitive, pleiotropic defects in receptor-mediated endocytosis. These mutants fall into the same two genetic complementation groups previously found with our non-conditional mutants. After transfer to non-permissive temperature maximal alteration of phenotype occurs within 3 hours. On return to permissive temperature normal function is restored in three steps: endosomal function, 4-6 hours; Golgi function, 6-8 hours; uptake of alpha2-macroglobulin and lysosomal hydrolases and compartmentalization of endogenous hydrolases, 24 hours. These last phenomena all involve ligands moving to lysosomes. We are now using the temperature-sensitive mutants: 1. to delineate the route to lysosomes taken by endogenous hydrolases; 2. to identify the proteins defective in the two classes of mutants; 3. as recipients in DNA-mediated transfection. We had hypothesized that the Golgi-associated defect in the endocytosis mutants resulted from their inability to acidify that organelle. But, no differences were found between mutants and parent with respect to accumulation of a weak base in the Golgi region. Continuing our studies on biosynthesis of asparagine-linked oligosaccharides, we have isolated a CHO cell mutant CHB11.1.3 which transfers a truncated Man 5 oligosaccharide, a structure found in mutants which cannot synthesize mannosyl phosphoryl dolichol. However, CHB11.1.3 accumulates mannosyl phosphoryl dolichol at levels 5 times those found in the parent, suggesting that it is defective in a mannosyl transferase. We have developed an in vitro assay for transferase activity.